This invention relates to an electrophoresis device for separating samples in a running gel, and more particulary to such a device for use in separating samples in running gels of any geometric configuration in one or two dimensions.
In the past severe problems have been encountered obtaining leak-tight seals for performing gel electrophoresis between glass plates and within glass tubes. For example, vertical slab gel electrophoresis is accomplished by using two square glass plates separated along the two vertical edges by spacers to define the gel thickness, and thereafter sealing the two sides and the bottom of the space between the plates with a liquid-tight tape. Thereafter, the space between the plates is filled with acrylamide or agarose and then polymerized into a gel state. The result is a separating or "running" slab gel between the glass plates. The tape along the bottom of the pair of plates is removed to expose the bottom of the slab gel. The glass/gel sandwich is then inserted into either a rectangular grommet or some other type of compression seal so that the top protrudes through the grommet into a top compartment which is isolated from a bottom compartment by the grommet or compression seal. The lower end of the gel is exposed to the bottom compartment. Upper and lower electrically conductive buffer solutions are deposited in the top and bottom compartments respectively so that when a DC voltage is impressed between the upper and lower buffer solutions an electrical field is set up between the upper and lower exposed edges of the running gel. A sample deposited on the top of the running gel is caused to migrate through the gel by the electric field, certain components of the sample being stopped at certain stratum within the gel as the sample is seemingly "sieved" by the running gel as it migrates therethrough. A major drawback to this type of apparatus and method is that leakage between the glass plates and the rectangular grommet or compression seal is difficult to eliminate and can only be overcome absolutely by additional sealing with grease or other substances. Moreover, the sides of the running gel are exposed through leakage past the liquid tight tape to the upper and lower buffer solutions causing spurious electrical fields to be set up along the sides of the running gel. This results in distortion of the sample separation. Apparatus and methods are known for eliminating the rectangular sealing grommet, but such apparatus and methods do not remove the side leakage problem and the consequent sample separation distortion. Such apparatus and method are described in copending U.S. Application Ser. No. 569,838 in common ownership with the apparatus and method disclosed herein.
Tube gel electrophoresis is also practiced wherein the running gel is formed within a tube and thereafter disposed with the upper and lower ends of the running gel exposed to upper and lower buffer solutions similar to those described above for the slab gel electrophoresis devices. The sealing problem is retained in the known tube gel electrophoresis apparatus and methods. Seals between the gel holding tubes and the structure through which the tubes must pass are required. The structure serves to isolate electrically the upper and lower buffer solutions. Seal leakage is a problem as well as sample distortion due to overheating resulting from the electrical energy dissapated within the running gels during electrophoresis.
Two-dimensional electrophoresis involves running a sample in one direction through a running gel to obtain a separation of the sample into strata, and thereafter performing electrophoresis in a direction substantially orthogonal to the first separation direction to obtain a number of substrata in the sample. This achieves a finer component separation for the sample. In the past, when two-dimensional electrophoresis was performed using a tube shaped running gel, the first electrophoretic separation was completed and the tube gel was removed from the tube. A slab gel having the same thickness as the diameter of the tube gel was formed between glass plates as described above, the tube gel laid across the top of the slab gel, and the sample strata in the tube gel electrophoresed downward out of the tube gel into the slab gel to obtain substrata separation therein. When attempting two-dimensional electrophoresis in a slab gel, the side seals were removed after the first electrophoretic separation in one direction through the slab gel, and the previously open ends were thereafter sealed. The slab was placed in contact with the upper and lower buffer solutions at the newly exposed sides of the slab gel. Subsequent electrophoretic separation provided sample component substrata as described hereinbefore. However, the usual risk of sample distortion due to leakage and spurious electrical fields was compounded by the breaking and reforming of seals at the edges of the slab gel.
Consequently, a device and method are needed for performing either one or two dimensional electrophoresis wherein the sealing problem and distortion from spurious electric fields and lingering heat of electrophoresis is substantially reduced.